Yonsei Med J.  2018 Nov;59(9):1079-1087. 10.3349/ymj.2018.59.9.1079.

Effects of Antioxidant Tempol on Systematic Inflammation and Endothelial Apoptosis in Emphysematous Rats Exposed to Intermittent Hypoxia

• Zhao H
• Zhao Y
• Li X
• Xu L
• Jiang F
• Hou W
• Dong L
• Cao J

• KMID: 2422493
• DOI: http://doi.org/10.3349/ymj.2018.59.9.1079

Abstract

PURPOSE
Obstructive sleep apnea and chronic obstructive pulmonary disease are independent risk factors of cardiovascular disease (CVD), and their coexistence is known as overlap syndrome (OS). Endothelial dysfunction is the initial stage of CVD; however, underlying mechanisms linking OS and CVD are not well understood. The aim of this study was to explore whether OS can lead to more severe inflammation and endothelial apoptosis by promoting endothelial dysfunction, and to assess the intervention effects of antioxidant tempol.
MATERIALS AND METHODS
Male Wistar rats (n=66) were exposed to normal oxygen [normal control (NC) group], intermittent hypoxia (IH group), cigarette smoke (CH group), as well as cigarette smoke and IH (OS group). Tempol intervention was assessed in OS group treated with tempol (OST group) or NaCl (OSN group). After an 8-week challenge, lung tissues, serum, and fresh blood were harvested for analysis of endothelial markers and apoptosis.
RESULTS
The levels of intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, and apoptosis in circulating epithelial cells were the highest in OS group and the lowest in NC group. These levels were all greater in IH group than in CH group, and were lower in OST group than in OS and OSN groups (all p < 0.001).
CONCLUSION
Synergistic effects of IH with cigarette smoke-induced emphysema produce a greater inflammatory status and endothelial apoptosis. OS-related inflammation and endothelial cell apoptosis may play important roles in promoting cardiovascular dysfunction, and antioxidant tempol could achieve a partial protective effect.

Keyword

Overlap syndrome; intermittent hypoxia; emphysema; inflammation; endothelial dysfunction; antioxidant

MeSH Terms

Animals
Anoxia*
Apoptosis*
Cardiovascular Diseases
Emphysema
Endothelial Cells
Epithelial Cells
Humans
Inflammation*
Lung
Male
Oxygen
Pulmonary Disease, Chronic Obstructive
Rats*
Rats, Wistar
Risk Factors
Sleep Apnea, Obstructive
Smoke
Tobacco Products
Oxygen
Smoke

Figure

• 

  Fig. 1 Oxygen concentration-time curve.

• 

  Fig. 2 Histological photographs from different groups, stained with hematoxylin and eosin and captured with light microscopy at 40× magnification. (A) NC group (normal oxygen control). (B) IH group (intermittent hypoxia exposure). (C) CH group (cigarette smoke exposure). (D) OS group (cigarette smoke and IH exposure). (E) OS group treated with NaCl (OSN group). (F) OS group treated with tempol (OST group).

• 

  Fig. 3 Histopathological assessment of the lung. Rats were exposed to different conditions as follows: NC group (normal oxygen control), IH group (intermittent hypoxia exposure), CH group (cigarette smoke exposure), OS group (cigarette smoke and IH exposure), OSN group (OS group treated with NaCl), OST group (OS group treated with tempol). Data are presented as means±SD. (A) *p<0.001 vs. NC or IH, †p<0.001 vs. CH. (B) *p<0.001 vs. NC, †p<0.001 vs. OS, ‡p<0.001 vs. OSN (one-way ANOVA and Bonferroni post-hoc multiple comparisons). MAN, mean alveolar number; MLI, mean linear intercept.

• 

  Fig. 4 Serum levels of cell adhesion molecules. Serum levels intracellular adhesion molecule-1 (ICAM-1) (A) and vascular cellular adhesion molecule-1 (VCAM-1) (B) were measured in rats from different groups (n=8 each). Data are presented as means±SD. Rats were exposed to different conditions as follows: NC group (normal oxygen control), IH group (intermittent hypoxia exposure), CH group (cigarette smoke exposure), OS group (cigarette smoke and IH exposure). *p<0.001 vs. NC, †p<0.001 vs. CH, ‡p<0.001 vs. IH (one-way ANOVA and Bonferroni post-hoc multiple comparisons).

• 

  Fig. 5 Flow cytometry analysis for CECs. (A) Apoptotic cells (Annexin V+ and 7-ADD−) are located in lower right quadrant (Q4). Lower left quadrant (Q3) contains healthy cells (Annexin V− and 7-AAD−). Upper right quadrant (Q2) contains late apoptotic and dead cells (Annexin V− and 7-ADD−). Damaged cells (Annexin V− and 7-AAD−) are displayed in lower right quadrant (Q1). (B) Quantification of the apoptotic rate of CECs in groups exposed to different conditions. Data are reported as mean±SD (*p<0.001 vs. NC, †p<0.001 vs. CH, ‡p<0.001 vs. IH; one-way ANOVA and Bonferroni post-hoc multiple comparisons). CEC, circulating endothelial cell; 7-ADD, 7-Amino Actinomycin D; NC, normal oxygen control group; IH, intermittent hypoxia exposure group; CH, cigarette smoke exposure group; OS, cigarette smoke and IH exposure group; OSN, OS group treated with NaCl; OST, OS group treated with tempol.

• 

  Fig. 6 Changes in serum levels of cell adhesion molecules and endothelial apoptosis with tempol treatment. Rats were exposed to IH together with smoke in OS group. In addition, animals were treated with 100 mg 10% (w/v) tempol (OST group) or 0.9% NaCl (OSN group) per kilogram of body weight by intraperitoneal injection before OS exposure daily. ICAM-1 (A), VCAM-1 (B), and CECs (D) were measured in animals (n=8 per group) and compared to rats from NC group. Flow cytometry analysis for circulating CECs (C) are presented from NC, OS, OSN, and OST groups. Data of (A), (B), and (D) are expressed as means±SD. *p<0.001 vs. NC, †p<0.001 vs. OS, ‡p<0.001 vs. OSN (one-way ANOVA and Bonferroni post-hoc multiple comparisons). CEC, circulating endothelial cell; ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cellular adhesion molecule-1; NC, normal oxygen control group; IH, intermittent hypoxia exposure group; CH, cigarette smoke exposure group; OS, cigarette smoke and IH exposure group; OSN, OS group treated with NaCl; OST, OS group treated with tempol.

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